Alkyl iodoacetal

The different configurations of the salts obtained by var3dng the sequence of alkylation are well illustrated by the reaction of pseudo-tropine (14) with ethyl iodoacetate to give 15, while the opposite order... 

Ribosomal protein L12 was oxidized with 0.3 M H202 at 30°C for 1 h. After dialysis, the protein was incubated in the presence of 0.8 M 2-mercaptoethanol for 48 min at 37 °C and dialyzed. The amount of methionine residues was quantitated by exhaustive alkylation of the protein with [14C]iodoacetic acid. 

An affinity label is a molecule that contains a functionality that is chemically reactive and will therefore form a covalent bond with other molecules containing a complementary functionality. Generally, affinity labels contain electrophilic functionalities that form covalent bonds with protein nucleophiles, leading to protein alkylation or protein acylation. In some cases affinity labels interact selectively with specific amino acid side chains, and this feature of the molecule can make them useful reagents for defining the importance of certain amino acid types in enzyme function. For example, iodoacetate and A-ethyl maleimide are two compounds that selectively modify the sulfur atom of cysteine side chains. These compounds can therefore be used to test the functional importance of cysteine residues for an enzyme s activity. This topic is covered in more detail below in Section 8.4. 

Figure 1.87 Iodoacetate can modify a number of amino acid side chains in proteins, forming alkylated derivatives containing a terminal carboxylate.

Another approach uses reactive alkyl halogen compounds containing a terminal carboxylate group on the other end to form spacer arms off the dextran polymer from each available hydroxyl. In this manner, Brunswick et al. (1988) used chloroacetic acid to modify the hydroxyl groups to form the carboxymethyl derivative. The carboxylates then were aminated with ethylene diamine to create an amine-terminal derivative (Inman, 1985). Finally, the amines were modified with iodoacetate to form a sulfhydryl-reactive polymer (Figure 25.14). 

Butenolides.1 When activated by silver trifluoroacetate, this furan is alkylated by primary alkyl iodides or ethyl a-iodoacetate to give 4-alkyl-2-butenolides (2) in 60-80% yield. 

The basis of the action of iodoacetate on muscle contraction was uncovered by Dickens and Rapkine (ca. 1933). They found iodoacetate alkylated SH groups on proteins, especially those in glyceraldehyde 3-phosphate dehydrogenase (G-3-PDH). When the enzyme was inhibited precursors accumulated—hexose mono- and diphosphates—as in Lundsgaard s experiments. 

FIGURE 4 Effect of sample preparation on the fragmentation of an rMAb observed in (A) SDS-PAGE and (B) CE-SDS with LIF detection. SDS-PAGE lanes (Lane I) molecular weight standards bovine serum albumin at (Lane 2) 8 ng and (Lane 3) 2 ng (Lane 4) rMAb control after alkylation with (Lane 5) iodoacetic acid and (Lane 6) iodoacetamide. (See color plate 4.)... 

More recently, Kim and coworkers have developed a novel radical alkylation reaction of organic nitro derivatives 16a-d via bis(silyloxy)enamines 17a-d (Scheme 16). This method enables not only P -alkylation to the nitro gronp, bnt also the conversion of the nitro group (16a-d) into an oxime ether fnnctionahty (18a-d). The irradiation of a solntion of 16a-d with iodomethyl phenyl snlfone (or ethyl iodoacetate) and hexamethylditin in benzene at 300 nm give the oxime ethers 18a-d in good yields. 

Tin-based reagents are not always snitable owing to the toxicity of organotin derivatives and the difficulties often encountered in removing tin residues from the final product. Therefore, the same authors have carried out additional experiments with 17d and several different alkyl halides under tin-free conditions. The treatment of 16d with tert-butyldiphenylsilyl chloride (TBDPSCl) and triethylamine in the presence of silver triflate in CH2CI2 affords the bis(silyloxy)enamine 17d in 92% yield (Scheme 17). When the radical reaction was carried out with ethyl iodoacetate in the presence of 2,2 -azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70) as the initiator in CH2CI2, the oxime ether 19 was obtained in 83% yield (Scheme 17). 

Under these reducing conditions of hydrolysis of tryptophan peptides, cystine is reduced to cysteine and its coelution with proline using standard buffer gradients, makes quantitation difficult. Thus, cysteine and cystine are generally derivatized prior to acid hydrolysis by oxidation to cysteic acid with performic acid 21 or alkylation, upon reduction in the case of cystine, with iodoacetic acid 21 or, more appropriately, with 4-vmylpyridine)22 23 50 Conversion of cysteine into 5- 3-(4-pyridylethyl)cysteine bears the additional advantage of suppressing epimerization via the thiazoline intermediate, thus allowing for standardization of the acid-hydrolysis dependent racemization of cysteine in synthetic peptides)24 ... 

In an extension of atom-transfer radical reactions to heterocyclic systems, Byers has introduced a novel methodology for the addition of electron-deficient radicals to unprotected pyrroles and indoles in a stannane-fi ee, non-oxidative process <99TL2677>. For exanqrle, photochemical reaction of pyrrole (33) with etl l iodoacetate (34) in presence of thiosulfiite as an iodine reductant, phase transfer catalyst and propylene oxide led to high yields of the 2-alkylated pyrrole 35 <99TL2677>. 

The iminium ions produced by the C- alkylation of A2-piperideines can have synthetic utility for the formation of additional carbon-carbon bonds. This concept is illustrated (Scheme 12) by the synthesis of vincamone and its epimer from piperideine (120). Treatment of enamine (120) with ethyl iodoacetate gave iminium ion (121) which cyclized to (122) under the reaction conditions. Completion of the synthesis was accomplished by base followed by acid treatment (82TL177). 

The surest way to inhibit an enzyme is to block the active site irreversibly by chemical reaction with some active species to form a covalent bond. Thus, iodoacetate will irreversibly inactivate thiol proteases by forming the stable carboxymethyl mercaptan. lodoacetate is of course non-selective (many other enzymes would be inactivated), toxic (many sensitive sites would be alkylated) and moreover the drug itself is unstable due to its very reactivity. 

In addition to the alkylation with iodoacetate (Eq. 3-24), sulfhydryl groups can react with N-ethylmale-imide (Eq. 3-39).281 This reaction blocks the SH groups irreversibly and has often been used in attempts to establish whether or not a thiol group plays a role in the functioning of a protein. Loss of function in the... 

Imidazole also acts as a substrate-competitive inhibitor, forming both binary complexes with LADH, and ternary complexes in the presence of coenzyme. X-Ray studies show that imidazole also binds to the. catalytic zinc by displacing the water molecule.1361 The presence of imidazole at the active site also enhances the rate of carboxymethylation14658 of Cys-46 with both iodoacetate and iodoacetamide.1420 This enhancement of alkylation has become known as the promotion effect .1421 Imidazole promotion also improves the specificity of the alkylation.1422 Since Cys-46 is thought to be alkylated as a metal-thiol complex, imidazole, on binding the active site metal, could enhance the reactivity by donating a electrons to the metal atom, which distributes the increased electron density further to the other ligands in the coordination sphere. The increased nucleophilicity of the sulfur results in promoted alkylation.1409... 

Price et al. (86). The evidence is based on experiments in which DNase I was reacted with iodoacetate at pH 7.2 in the presence of 0.1 M Mn2+. Under these conditions the enzyme is gradually inactivated and the loss of activity parallels the formation of one residue of 3-carboxymethyl histidine per molecule. The rate of the alkylation reaction is dependent on Mn2+ concentration. Substitution of Mn2 by Cu2+ in the presence of tris buffer greatly increases the rate of alkylation. A 29-residue peptide con-... 

The investigations of W. H. Stein and Moore and their colleagues were first reported in 1959 157). The inactivation of RNase by iodo-acetate was studied. A maximum in the rate of activity loss was noted at pH 5.5. Reaction with a methionine residue was found at pH 2.8 at pH 8.5-10 lysine residues were modified, but at pH 5.5-6.0 only histidine appeared to be involved. The specific reaction required the structure of the native enzyme. Reaction with histidine was not observed under a variety of denaturing conditions 158). Iodoacetamide did not cause activity loss, or only very slow loss, or alkylate His 119 in the native enzyme at pH 5.5. The negative charge on the carboxyl group of the iodoacetate ion was apparently essential. 

Alkylation at pH 8.5 shows reduced rates of reaction at the histidine residues but significant substitution at lysine, particularly Lys 41 118). The histidine reactions show the same general stereospecificity as found at pH 5.5. The inactive Lys 41 derivatives (25, 26, and 27 of Table VI) show alkylation patterns of His 12 and 119 at pH 5.5 which are similar to those of RNase-A although with some differences in detail. When Lys 1 and 7 are acetylated in RNase-S the alkylation pattern with iodoacetic acid is not affected. When PIR is used the alkylation of His 119 is nearly abolished but that at His 12 is accelerated 163). The probable interaction of Asp 121 with His 119 may be important in the alkylation reactions observed in the native enzyme and the various lysine derivatives. In PIR this interaction has, of course, been removed. 

The probable general structure of the dimers was established in elegant experiments by Fruchter and Crestfield 381) involving alkylation with iodoacetate. The two isomeric dimers referred to above behave identically in these reactions. The two active sites in the dimers behave just like that of the monomer. Histidines 12 and 119 both react, but the reactions are mutually exclusive. The proposed structure is outlined in Fig. 19. The tail of one monomer combines with the body of the other and vice versa. The His 12 and 119 pairs are now on separate molecules. When the dimers, fully inactivated by reaction with iodoacetate, are dissociated by heating at neutral pH, the following monomers would be expected native RNase (active), CM-His-12-RNase (inactive) CM-His-119-RNase (inactive), and di-CM-His-12-His-119-RNase (inactive). These were, in fact, found. About 2b% activity reappeared from the inactive dimer. Equally important the di-CM compound was found. This material... 

Antigen unmasking on sections of paraffin-embedded tissues can be accomplished by reduction of disulfide bonds by treatment with 2-mercaptoethanol, followed by alkylation with sodium iodoacetate to prevent the bonds from reforming. This method has been used for unmasking a Kunitz protease inhibitory domain epitope of Alzheimer s amyloid precursor protein in human brain (Campbell et al., 1999). Sections are reduced with a mixture of 0.14 M 2-mercaptoethanol in 0.5 M Tris-HCl (pH 8.0) and 1 mM EDTA for 3 hr in the dark at room temperature. After being washed for 3 min in distilled water, the sections are treated with a mixture of 250 mg/ml iodoacetic acid in 0.1 M NaOH, diluted 1 10 in 0.5 M Tris-HCl (pH 8.0) and 1 mM EDTA for 20 min in the dark. 

Figure 2. Tryptic map of human relaxin B-chain. The peptide was reduced with dithiotreitol and alkylated with iodoacetic acid before digestion with trypsin. The chromatography was performed on a Vydac Cis column using TFA-containing mobile phases, and eluted with an acetonitrile linear gradient.

In the previous sections, the reactions of nucleophilic alkyl and acyl radicals with electron-deficient aromatics via SOMO-LUMO interaction have been described. At this point, we introduce the reactions of electrophilic alkyl radicals and electron-rich aromatics via SOMO-HOMO interaction, though the study is quite limited. Treatment of ethyl iodoacetate with triethylborane in the presence of electron-rich aromatics (36) such as pyrrole, thiophene, furan, etc. produces the corresponding ethyl arylacetates (37) [50-54]. 

Lovenberg, Buchanan, and Rabinowitz found that treatment of ferredoxin with iodoacetate or N-ethylmaleimide in either the presence or absence of 8 M urea had no effect on its spectral characteristics. Less than 1 mole of carboxymethyl cysteine was formed per mole of protein when native ferredoxin was treated with iodoacetate-1-C14 (Table 10). Sobel and Lovenberg (96) showed recently that C14-iodoacetate did not react appreciably with reduced ferredoxin. However, Table 10 shows that if ferredoxin was treated with 2-mercaptoethanol in 8 M urea, it was alkylated with iodoacetate. This demonstrated that the half-cystine residues of native ferredoxin were not present as free sulfhydryls, and the mercurial titration data given above showed that they were not present as disulfides. The two observations were consistent, therefore, with a structure in which the half-cystine residues are present as cysteine and are bonded with the iron by a sulfide bridge. 

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